Expression of sex-specific molecular markers by Babesia bovis gametes

Background Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. Methods and results We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. Conclusions Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis. Graphical Abstract


Background
Bovine babesiosis, caused by the intraerythrocytic protozoan parasite Babesia bovis, is an acute tick-borne hemolytic disease that affects cattle throughout the world [1,2].Babesia parasites are transmitted by the one-host tick Rhipicephalus microplus [3].Transmission of Babesia parasites from the bovine host to the tick vector requires the formation and development of parasite sexual stages inside the tick midgut [4].Inside the tick midgut, morphologically distinct male and female B. bovis gametes [5] mate and fuse to form zygotes. Zygotes develop into the kinete stage that circulates in tick hemolymph [6].After kinete invasion of eggs, the parasites are transmitted transovarially [4] resulting in larval progeny containing B. bovis sporozoites that can infect cattle [4].Bovine babesiosis is poorly controlled, and new vaccines are urgently needed [7].Vaccines targeting blood stages of Babesia parasites have proven to be largely ineffective [7].Transmission blocking vaccines may provide alternative strategies to control the spread of Babesia.However, transmission blocking vaccine development is currently limited by our poor understanding of parasite biology in the tick, especially at the molecular level [7].To date, there are no methods to isolate B. bovis sexual stages from infected tick midgut.We induced B. bovis sexual stage development in in vitro cultures using xanthurenic acid (XA), an intermediate metabolite derived from tryptophan metabolism [8,9].In vitro induction of B. bovis sexual stages allowed identifying sexual stage-specific genes and gene families [9][10][11].In this study, we examined the gene expression of B. bovis sex-specific markers for male and female gametes.Examined genes were selected based on previously identified gamete-specific genes in Plasmodium falciparum [12][13][14].The corresponding proteins in Babesia parasites are conserved and related to the development of sexual stages among all Apicomplexan parasites [15].Six target genes, including cyclic adenosine 3' ,5'-monophosphate (cyclic AMP)-dependent protein kinase cAMPDPK (pka), Hapless2/gcs1 (hap2), α-tubulin I, α-tubulin II, zinc finger C3H1 protein2 (znfp2), and ABC transporter and two gene families, thrombospondin-related anonymous proteins (trap1-4) and LCCL domain-containing proteins (ccp1-3), were selected.In the present study, we tested the expression patterns of B. bovis target genes and gene families in male gametes and in female-enriched gametes to identify sex-specific molecular markers for B. bovis male and female gametes.

Cattle and parasite cultures
A splenectomized 3-month-old male Holstein calf tested negative for B. bovis by PCR [4], and cELISA [18] was used in this study.The calf was inoculated intravenously with B. bovis S 74 -T 3 Bo strain stabilate containing approximately 1 × 10 7 B. bovis-infected erythrocytes [4,6].The infected calf was monitored daily for B. bovis in peripheral blood and clinical signs of babesiosis.The animal was maintained according to protocols approved by the University of Idaho Institutional Animal Care and Use Committee (IACUC #2018-16).

Babesia bovis blood stages
Defibrinated blood was collected from the calf at 11 days post-B.bovis inoculation.Blood was collected into flasks containing glass beads and shaken to prevent blood coagulation.Red blood cells from defibrinated blood were washed with Puck's Saline G to remove white blood cells.Some washed infected RBCs were pelleted by centrifugation at 500 ×g, 10 min at 4 °C, and suspended in TRIzol (ThermoFisher Scientific, Waltham, MA, USA).To establish B. bovis in vitro culture, infected RBCs were placed into flasks with culture medium as previously described [19] and incubated at 3% O 2 and 5% CO 2 .After the in vitro incubation of B. bovis blood stages, B. bovis cultures were used to induce sexual stages.

In vitro induction of B. bovis sexual stages
To induce sexual stages, in vitro cultured B. bovisinfected erythrocytes with 10% PPE were suspended in a medium with 100 μM XA (Sigma, St. Louis, MO, USA) at 26 °C with 5% CO 2 as previously described [9].Induced in vitro sexual stage parasites were isolated at 24 h postinduction by differential centrifugation at 400 × g for 1 min.

Separation of B. bovis male gametes
Male gametes were allowed to swim from the pellet of induced parasites that contained extracellular male and female gametes by incubation at room temperature for 15 min.The supernatant containing male gametes was harvested and spun at 12,000 ×g for 10 min at 4 °C and washed twice in phosphate buffered saline (PBS).The purity of the isolated males was determined by microscopy to be at 94%.Slides were stained using the Hema 3 Stat Pack (Thermo Fisher Scientific, Inc., Waltham, MA, USA).A portion of the male gametes and femaleenriched gametes was suspended in TRIzol and stored at − 20 °C.Another portion of the parasites was used for immunofluorescence assays.

RNA extraction and cDNA synthesis
Total RNA was extracted from B. bovis blood stages, male and female-enriched gametes collected from in vitro induced parasites in TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol and RNA pellets suspended in 20 µl DEPCtreated water (Invitrogen, Waltham, MA, USA).RNA samples were treated with DNase I (Invitrogen, Waltham, MA, USA) following the manufacturer's protocol to remove contaminating genomic DNA and quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA).The removal of genomic DNA was confirmed by PCR targeting rap1 as previously described [20] using non-reverse transcribed samples.cDNA was synthesized from 150 ng of total RNA of each sample with a Superscript ® First-strand cDNA synthesis kit (Invitrogen, Waltham, MA, USA) following the manufacturer's protocol.

Quantitative PCR assay
Specific primers for each gene were designed using the PrimerQuest ® Tool (Integrated DNA Technologies) (Table 1) following recommended guidelines for qPCR primer design.Standard PCR was performed to amplify all target genes from cDNA samples using primers listed in Table 1.PCR cycling conditions consisted of 95 °C for 3 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension of 72 °C for 5 min.PCR products were visualized by 1% agarose gel electrophoresis.PCR amplicons were cloned into PCR 2.1-TOPO ® (Thermo Fisher Scientific) and submitted for sequencing (Eurofins MWG Operon, Louisville, KY).Standard curves were generated for each gene using specific quantities of each plasmid.For the normalization of qPCR data, B. bovis actin (BBOV_IV009790) was used as a reference gene candidate [11].CFX Manager ™ software (Bio-Rad, Hercules, CA, USA) [21] was used to examine the stability of expression of the reference gene.The qPCRs for the genes of interest and reference gene were performed in a CFX96 ™ Real-Time PCR Detection System (C1000 Touch ™ Thermal Cycler) (Bio-Rad, Hercules, CA, USA) using the SsoFast ™ EvaGreen ® Supermix Kit (Bio-Rad, Hercules, CA, USA).The cycling conditions consisted of an initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C denaturation for 15 s and annealing at 55 °C for 30 s.Reactions were performed in triplicate in 20 μl using 300 nM of each primer and 2 μl of 1/20 dilution of cDNA as template.CFX Manager ™ software (Bio-Rad, Hercules, CA, USA) was used to analyze the qPCR data.Relative expression was calculated by dividing each gene's detected expression by the detected actin expression within each life stage.Pairwise differences of stages were tested with Tukey or Tukey-Kramer adjustment.

Antibody production
Polyclonal antibodies against HAP2 and TRAP1-4 were produced as described previously [9,22] by immunizing rabbits with synthetic peptides.For each protein, three synthetic peptides predicted to be surface-exposed B-cell epitopes using a proprietary algorithm were synthesized (BioSynthesis, Inc.Lewisville, TX, USA) and used to immunize rabbits (Table 2).

Detection of B. bovis surface exposed proteins on male and female gametes
A transfected B. bovis line tfBbo5480 expressing eGFP [23] was used to generate sexual stages for immunofluorescence assay (IFA).Live B. bovis male and femaleenriched gametes separated from induced sexual stage cultures were washed in PBS plus 3% bovine serum albumin (BSA).Cells were then incubated for 1 h with a 1:100 dilution of primary antibodies (anti-HAP2 or anti-TRAP1-4) in blocking solution.The cells were then washed twice in PBS by 400 ×g centrifugation and incubated for 30 min with 1:1000 goat anti-rabbit IgG Alexa

In silico analysis of target genes
In silico analysis was performed to select B. bovis homologs to previously identified P. falciparum male and female gamete-specific proteins.In silico predictions suggested the presence of a signal peptide in B. bovis HAP2, CCP1, TRAP1-4 proteins and transmembrane domains in B. bovis HAP2, ABC transporter and TRAP1-4 proteins.Babesia bovis target proteins shared from 22 to 90% amino acid identity with their orthologous proteins from P. falciparum (Table 3).Overall, B. bovis proteins appeared well conserved compared with homologs in P. falciparum.Babesia bovis target protein IDs and functional annotation are shown (Table 3).

Quantitative PCR and gene expression
Quantitative PCR was used to analyze the transcription pattern of target genes and gene families in B. bovis male gametes and female-enriched gametes collected from in vitro induced sexual stage parasites as well as blood stages.The transcription levels of all target genes were normalized to B. bovis actin expression level.The data demonstrated that pka, hap2, α-tubulin II and znfp2 were significantly upregulated in male gametes compared to female-enriched gamete samples or blood stages (P < 0.001) (Fig. 1).However, α-tubulin I and ABC transporter (Fig. 1), trap2-4 (Fig. 2) and ccp1-3 genes (Fig. 3) were significantly downregulated in male gametes compared to female-enriched gamete samples or blood stages (P < 0.05).

Discussion
Recently, we described the induction of B. bovis sexual stages in vitro using Babesia culture recovered from an acutely infected animal.The induction method yields high numbers of clean sexual stages, free of tick antigens.differentiation into large spherical shapes (female gametes) and a smaller shape that displayed one or more projections (male gametes) [9].Male gametes have up to 10 flagella randomly distributed around the cell.Swimming movement of B. bovis male gametes allowed us to separate male gametes for further analysis.In addition, we demonstrated that pka, hap2, α-tubulin II and znfp2 genes were upregulated in B. bovis male gametes.We also showed that α-tubulin I and ABC transporter were upregulated in female-enriched B. bovis gametes.Our data are consistent with previous studies in P. falciparum that showed genes were differentially expressed in male and female gametocytes during sexual development including pka, hap2, α-tubulin 2, znfp2, α-tubulin I and ABC transporter [12,14,[24][25][26].PKA has several functions in the cell, including regulation of glycogen, sugar and lipid metabolism.PfPKA is a key regulator of P. falciparum development [27].In P. falciparum, PKA is a male gamete-specific gene [12].HAP2 of Plasmodium is a protein known to be on the surface of microgametes (male gamete-specific protein) and HAP2-mediated gamete fusion in P. falciparum [28].HAP2 in B. bovis and B. bigemina were found to be expressed by in vitro induced sexual stages and during the development of Babesia parasites in the tick midgut [9,29,30].In Plasmodium, α-tubulin-2 is expressed abundantly by the male gametocytes but is present at low levels in female gametocytes or ookinetes [25,30].Alpha-tubulin 2 was found to be highly expressed in P. falciparum male gametocytes and form microtubules of the axoneme of male gametes [25].ZNFP2:CCCH-type zinc finger proteins are involved in RNA stability and transcriptional repression [31,32] functioning in both DNA and RNA regulation [33].Babesia bovis ZNFP2 is an ortholog to P. falciparum CCCH-ZNF4, which is important for exflagellation of gametocytes [33], while α-tubulin 1 is expressed in female gametes and ookinetes [26].Antibodies against P. falciparum α-tubulin-1 inhibited P. falciparum oocyst development in mosquito midguts [26].The genome of the B. bovis parasite encodes multiple members of ATPbinding cassette (ABC) transporters [15], one of which is an ortholog to P. falciparum gABCG2, which is transcribed predominantly by the female gametocyte [24].
Additionally, we demonstrated that trap2-4 and ccp1-3 genes were upregulated in female-enriched gametes in agreement with the previous P. falciparum RNA seq data [12], which showed TRAP and CCp families were upregulated in female gametocytes.The TRAP family [15] contains the vWFA and TSP-1 domains, which assist in erythrocyte invasion [34][35][36].Babesia bovis TRAP1-4s were recommended to be potential candidates for vaccine development [15,22].Members of the widely conserved CCp family, CCp1-3, are multidomain adhesion proteins containing LCCL motifs.CCp1-3s are differentially expressed on gametocytes of apicomplexans, including Plasmodium spp.and Babesia spp.[10,37,38], and are expressed on female gametes of P. falciparum [12].Knocking out ccp genes in P. falciparum led to the blocking of sexual stage development of the parasite in the mosquito vector [37,39].Members of the CCp family have been previously identified in Babesia bovis [10], B. bigemina [38] and B. ovata [40] as adhesion proteins and expressed upon gamete induction.Here, we demonstrate the expression of HAP2 and TRAP2-4 proteins on the surface of male and female-enriched gametes, respectively, consistent with our previous studies performed with B. bovis that demonstrated HAP2 [9] and TRAP2-4 [22] were expressed on in vitro induced sexual stages.The qPCR and IFA data confirm the lack of expression of B. bovis TRAP1 in either male or female gametes, which agrees with our previous study [22] that demonstrated expression of TRAP1 in B. bovis kinetes only with no expression by induced sexual stages.

Conclusions
In this study, we document that pka, hap2, α-tubulin II and znfp2 are male gamete-specific molecular markers and α-tubulin I, ABC transporter, trap2-4 and ccp1-3 are molecular markers for female gametes.In addition, HAP2 and TRAP2-4 proteins were found to be expressed on the surface of male and female gametes, respectively.The possible involvement of these proteins in the mechanism of gamete maturation and fusion strongly suggests their future use as potential candidates for developing Babesia transmission blocking vaccines.

Fig. 1
Fig.1Transcriptional analysis of the Babesia bovis pka, α-tubulin II, hap2, znfp2, α-tubulin I and ABC transporter genes in blood stages and male and female-enriched gametes.The data represent the mean of three experiments, each containing three technical replicates.Asterisk (**) indicates statistical pairwise differences (P < 0.001)

Fig. 2
Fig.2Transcriptional analysis of the Babesia bovis trap1-4 genes in blood stages and male and female-enriched gametes.The data represent the mean of three experiments, each containing three technical replicates.Asterisk (*) indicates statistical pairwise differences (P < 0.05)

Fig. 3 Fig. 4
Fig. 3 Transcriptional analysis of the Babesia bovis ccp1-3 genes in blood stages and male and female-enriched gametes.The data represent the mean of three experiments, each containing three technical replicates.Asterisk (*) indicates statistical pairwise differences (P < 0.05)

Table 1
Gene identification, primer sets of Babesia bovis genes of interest used for quantitative RT-PCR

Table 2
Peptides used for rabbit immunization to generate specific antibodies against Babesia bovis HAP2 and TRAP1-4 proteins

Table 3
The protein sequence identities (%) and domains of Babesia bovis homologs to Plasmodium falciparum target proteins SP signal peptide, TM transmembrane domain